ABSTRACT
Functional expression of bovine visual rhodopsin in the cell-free translation system with cotranslational insertion of the protein into phosphatidylcholine liposomes is described. The recombinant rhodopsin has spectral and functional properties similar to those of natural rhodopsin from bovine retina. Two mutant rhodopsins with amino acid substitutions in the hydrophilic C-terminal domain were obtained using oligonucleotide-directed mutagenesis. It was found that substitution Cys-316----Ser does not affect rhodopsin's ability to activate the visual amplification cascade, whereas double mutation Asp-330----Asn, Asp-331----Asn dramatically lowers the rhodopsin functional activity.
Subject(s)
Mutation , Protein Biosynthesis , Retinal Pigments/biosynthesis , Rhodopsin/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cell-Free System , GTP Phosphohydrolases/metabolism , Kinetics , Molecular Sequence Data , Oligonucleotide Probes , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodopsin/genetics , Rhodopsin/metabolism , Transducin/metabolismABSTRACT
Influence of structural changes in nontranslated regions and translation initiation site of the in vitro synthesized bovine opsin mRNA on its translational efficiency in the wheat germ cell-free system has been studied. It is shown that level of the opsin synthesis up to 30 micrograms per 1 ml of translational mixture can be attained by optimizing structure of 5'-nontranslated region.
Subject(s)
Eye Proteins/genetics , Protein Biosynthesis , RNA, Messenger/genetics , Retinal Pigments/biosynthesis , Retinal Pigments/genetics , Rhodopsin/biosynthesis , Animals , Base Sequence , Cattle , Cell-Free System , Cloning, Molecular , DNA/genetics , Genetic Vectors , Molecular Sequence Data , Mutation , Oligonucleotides/genetics , Plasmids , Precipitin Tests , Rod Opsins , Transcription, GeneticABSTRACT
The complete primary structure (1449 b. p.) of mobile genetic element ISH S1 from Halobacterium halobium has been elucidated using the dideoxy/M13 sequencing procedure. Computer analysis of the structure reveals similarity in overall structural organization of ISH S1 and other known transposable genetic elements of halobacteria and makes it possible to propose a hypothetical model of halobacterial promoter.
